The trillions of bacteria that make up the human microbiome are believed to encode functions that are important to human health; however, little is known about the specific effectors that commensal bacteria use to interact with the human host. The inability to culture many commensal bacteria renders these microbes incompatible with the most heavily relied upon techniques for characterizing effector molecules. To solve this problem requires the application of methods where bacterial effectors can be isolated and observed to interact with human biology. In functional metagenomic studies fragments of DNA extracted from an environmental sample are cloned into a model bacterial host, and the resulting metagenomic clones are examined for phenotypes of interest. This approach circumvents the culture barrier allowing for the simultaneous identification of effectors from both cultured and uncultured microbes. In a recently submitted manuscript we demonstrated the use of functional metagenomic techniques to isolate commensal effector molecules. In this study we created three metagenomic libraries from DNA isolated from phenotypically diverse patient stool samples. High content imaging of a human reporter cell line was used to identify effector molecules produced by metagenomic clones that activate human cellular NF?B pathways. This study led to the discovery of 26 biosynthetic commensal effector genes and a small molecule, N-acyl- 3OH-palmitoyl-glycine which structurally mimics endogenous signaling molecules in humans and modulates immune cell functions. The central hypothesis is that our functional metagenomic screening method can be broadly applied to the human microbiome to discover effector molecules by expanding our metagenomic library collection and repertoire of human cellular reporters (Aim 1). Once metagenomic clones are isolated that interact with human cells it is straightforward to identify each effector gene and molecule (Aim 2) and explore the chemical diversity of similar molecules produced by other commensals (Aim 3). The rationale that underlies this proposal is that the isolation of bioactive molecules creates a strong foundation for future research to understand how commensal effector functions dictate host physiology and to target these host- microbial interactions for therapeutic development. To achieve these aims I will be supported by a primary mentor who is a scientific leader in the application of functional metagenomic research methods, Dr. Sean Brady (Rockefeller University) and a co- mentor with expertise in host-microbial interactions and mucosal immunology, Dr. Sergio Lira (Mount Sinai). Each mentor will help me complete the individual aims of this project and develop the skills required to pursue my long term goal to understand how commensal effector molecules define host-microbial interactions.